Purified AIDS-associated virus

ABSTRACT

A newly discovered family of AIDS-associated viruses, designated ARV, is described. The viruses were isolated from AIDS patients from San Francisco and (a) are type D retroviruses; (b) have Mg ++  --dependent reverse transcriptase activity; (c) induce human multinucleated cells without immortalizing the cells; (d) are replicable in HUT-78 human T cells; and (e) induce viral protein(s) in HUT-78 that binds to Ig from AIDS patients. The infected HUT-78 cells and immunogenic polypeptides derived from the viruses are useful for diagnosing AIDS.

REFERENCE TO GOVERNMENT GRANT

This invention was made with Government support under grant no. CA-34980awarded by the National Institutes of Health. The Government has certainrights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 07/956,682filed Oct. 5, 1992 now abandoned , which application is a continuationof application Ser. No. 07/628,500 filed Dec. 17, 1990 (now abandoned)which is a continuation of application Ser. No. 07/433,393 filed Nov.7,1989 (now abandoned) which is a continuation of application Ser. No.07/080,635 filed Jul. 31, 1987 (now abandoned) which is a divisionalapplication of application Ser. No. 06/641,167 filed Aug. 15, 1984 nowU.S. Pat. No. 4,716,102, to which applications is claimed priority under35 USC § 120.

DESCRIPTION

1. Technical Field

This invention relates to the diagnosis and treatment of acquired immunedeficiency syndrome (AIDS). More specifically it relates to a newlydiscovered family of AIDS-associated viruses and the use of thoseviruses or materials derived from them to diagnose and/or treat AIDS.

2. Background Art

AIDS is known to have affected thousands of individuals worldwide. It isprevalent particularly in populations of male homosexuals. Also manyinstances of unexplained chronic lymphadenopathy may be caused by thesame pathogenic agent that causes AIDS.

Other investigators have reported AIDS-associated viruses. Pope, J. W.,et al, N Engl J Med (1983) 309:945 and others have reported isolating ahuman retrovirus designated HTLV-I from AIDS patients. HTLV-I, which isassociated with human T cell leukemia, has a type C morphology byelectron microscopy (EM), can immortalize T cells, and is primarilycell-associated (Poiesz, B. J., et al, PNAS (1980) 77:7415).Barre-Sinoussi, F., Science (1983), Montagnier, J. C., et al, ColdSpring Harbor Symposium (1984) in press, and Vilmer, E., et al, Lancet(1984) i:753 have found another retrovirus, LAV, in AIDS patients. LAV,which was isolated initially from the lymph node of a patient withlymphadenopathy, has a type D morphology by EM, causes cytopathicchanges in T cells, and is infectious in culture fluids. Popovic, M. G.,et al, Science (1984) 224:497 and Gallo, R. C., et al, Science (1984)224:500 report a third retrovirus, HTLV-III, that is present in patientswith AIDS and related syndromes. HTLV-III shows some cross-reactivitywith the other HTLV viruses, but, like LAV, has a type D morphology byEM and causes cytopathic changes in lymphocytes.

The present invention concerns a family of AIDS-associated retrovirusesthat has been isolated from a population of homosexual AIDS patients inSan Francisco.

DISCLOSURE OF THE INVENTION

The newly discovered family of AIDS-associated viruses have beendesignated ARV. These viruses are believed to be similar to but distinctfrom LAV.

In one aspect the invention concerns purified AIDS-associatedretroviruses (ARV), which are characterized by:

(a) being of type D morphology by electron microscopy;

(b) having Mg⁺⁺ --dependent reverse transcriptase activity;

(c) being capable of inducing human multinucleated cells withoutimmortalizing the cells;

(d) being capable of replicating in HUT-78 human T cells; and

(e) inducing viral protein(s) in HUT-78 that is recognized byimmunoglobulins from AIDS patients.

A second aspect of the invention relates to immortal human T cell lines,preferably the human T cell line HUT-78, chronically infected with anARV virus, preferably ARV-2.

A third aspect of the invention concerns a method of detecting anti-ARVantibodies in a sample of an immunoglobulin-containing body fluid of apatient comprising:

(a) incubating at a pH of about 7.2 to about 7.4 the chronicallyinfected T cell line in a fixed form or an immobilized (solid-phase)immunogenic ARV polypeptide with the sample under conditions thatpromote antigen-antibody binding;

(b) thereafter separating the cells or solid phase from the body fluidand washing the cells or solid phase to remove any residual unbound bodyfluid therefrom;

(c) incubating the washed cells or solid phase with a labeled antibodyto human Ig under conditions that promote antibody-antigen binding;

(d) thereafter separating the cells or solid phase from the labeledantibody and washing the cells or solid phase to remove unbound labeledantibody therefrom; and

(e) detecting immune complexes that include said anti-ARV antibodies onor in the cells or solid phase via the label.

Other aspects of the invention concern kits for carrying out the aboveassay method, reverse-transcribed DNA of the genomic RNA of an ARVvirus, fragments of such DNA, labeled embodiments of the DNA or DNAfragments that are useful as DNA hybridization probes, DNA hybridizationand cytohybridization assays for detecting ARV virus that employ thelabeled DNA probes, and synthetic expression vectors and replicons thatinclude such DNA or fragments thereof that are useful for reproducingthe DNA and expressing the DNA in transformed host cells or organisms soas to produce ARV polypeptides.

Still other aspects of the invention relate to: methods of preparingantibodies to ARV that involve immunizing host animals with ARV; andAIDS vaccines whose immunogenic agent is an ARV polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a graph showing the Mg⁺⁺ --dependent reverse transcriptaseactivity of the virus detected in peripheral mononuclear cell (PMC)cultures; and

FIG. 2 is a graph of the sucrose gradient of the example, infra.

MODES FOR CARRYING OUT THE INVENTION

The retroviruses of the invention were obtained from PMC samples(primarily from patients in the early stages of disease) or bone marrowsamples of homosexual AIDS patients from San Francisco, Calif., USA. ThePMCs were obtained from heparinized blood by Ficoll-Hypaque gradientcentrifugation followed by washing. The PMCs were cultured by plating ina liquid growth medium (RPMI 1640 supplemented with 10% fetal calf serumand antibiotics) at 2×10⁶ cell/ml. Lymphocyte stimulaters such asphytohemagglutinin and IL-2, were added to the medium to stimulatelymphocyte proliferation. Polybrene (1 μg/ml) was added to enhance virusspread to fresh cells. After a week of culture surviving cells wereprimarily of T cell lineage. Virus-containing cultures were identifiedby screening culture supernatants for Mg⁺⁺ --dependent reversetranscriptase (RT) activity.

Cell free virions exhibiting such RT activity were isolated from thesupernatants by sucrose gradient centrifugation, with ARV particlesbanding at 1:14 to 1:16 g/ml.

CHARACTERIZATION OF VIRUSES

The isolated viruses induce multinucleated cells in lymphocyte cultureswithout immortalizing the cells. Morphology typing of the viruses by EMshowed them to be type D retroviruses. Budding forms showed earlyfeatures of both type C and type D retroviruses, but only mature type Dretroviruses were observed in the cultures. Cultures infected with theisolated viral particles reacted with sera from AIDS patients from SanFrancisco and elsewhere and with anti-LAV sera.

ESTABLISHMENT OF ARV VIRUSES IN CONTINUOUS CULTURE

The human T cell lines MOLT-4 (ATCC CRL 1582), CCRF-CEM (ATCC CCL 119),and HUT-78 (obtained from A. Gazdar, Bethesda, Md.) were cultured inRPMI 1640 supplemented with serum, antibiotics, polybrene and anti-humanalpha interferon. ARV were then added. The MOLT-4 and CCRF-CEM linescould not be infected successfully under these conditions but the HUT-78line replicated the viruses in substantial titer. The resultingchronically infected HUT-78 variants are useful for propagating theviruses, as a source of viral components (e.g., nucleic acids, envelopeproteins) and for use in immunoassays for detecting anti-ARV antibodiesin Ig-containing samples (typically sera) of patients and otherindividuals.

A sample of HUT-78 infected with an ARV designated ARV-2 (describedbelow) was deposited at the American Type Culture Collection, 12301Parklawn Drive, a Rockville, Md., USA, on 9 Aug. 1984 under accessionnumber CRL 8597. This deposit was made under the Budapest Treaty andwill be maintained and made available in accordance with the provisionsthereof.

IMMUNOASSAYS USING HUT-78 CELLS OR IMMOBILIZED ARV POLYPEPTIDES

In immunoassays for anti-ARV antibodies, infected HUT-78 cells orpolypeptides that define the immunogenic determinants of ARV immobilizedon a solid phase are incubated with a sample of the patient's plasma orserum that has been heat-treated (e.g., heated to 56° C. forapproximately 1/2hr) to inactivate complement. The HUT-78 cells arefixed beforehand such as by treatment with cold acetone for about 15 minfollowed by air drying or treatment with 3% paraformaldehyde. Theincubation is carried out under conditions that permit and promoteantigen-antibody binding. The incubation will typically be carried outat physiological temperature (˜37° C.), and a pH of 7.2 to 7.4,preferably 7.3. The incubation time is preferably about 1/2hr. After theincubation the cells or solid phase are separated from thesample-containing medium and washed to remove any unbound sample. Thecells or solid phase are then resuspended in a medium containing alabeled anti-human Ig antibody using the same or similar incubationconditions. After this second incubation, the cells or solid phase areseparated from the medium containing the labeled antibody and washed toremove unbound labeled antibody. The presence of immune complexes in/onthe cells or solid phase are detected via the label. The mode ofdetection will depend upon the nature of the label. Radionuclide labels,such as ³ H or ¹²⁵ I, will be detected by scintillation counting.Fluorochrome labels, such as a fluorescein isothio-cyanate, will bedetected by subjecting the cells or solid phase to excitation energy andobserving the resulting fluorescence using a fluorescent microscope.Enzyme labels, such as peroxidases, are detected by incubating the cellsor solid phase in a substrate-containing medium and detecting enzymeactivity through spectrophotometric measurements of the medium. Otherlabels may be used as is known in the art.

Kits for carrying out the above-described assay contain the followingcomponents: fixed cells on a support such as a slide or cover slip orimmunogenic ARV polypeptide bound to a solid phase, such as a cellulosederivative, polystyrene, beaded agarose, cross-linked dextrans, glass,and the like; incubation medium buffered to the desired pH; positive andnegative antibody controls; and labeled anti-human Ig antibody. The kitswill also typically contain instructions and, in the case of enzymeassays, substrates. Conventional techniques for packaging the kit andits components may be used.

DNA DERIVED FROM ARV

ARV-derived polynucleic acid for use in making probes may be obtainedfrom ARV particles or chronically infected HUT-78 cells. Nucleic acidsare liberated from the particles or cells, isolated, and digested withone or more restriction enzymes to produce nucleic acid fragments ofappropriate size. Particular fragments may be isolated from the digestby gel electrophoresis, if desired. The desired fragments may bereplicated by cloning them into a suitable vector (prokaryotic oreukaryotic) to form a replicon. A suitable host is then transformed withthe replicon and transformants are propagated to produce copies of thevirus-derived nucleic acid fragment. The fragments may be recovered fromthe host by extracting nucleic acids from the transformants, restrictingthe nucleic acids with the same endonuclease(s) used previously andresolving the digest, such as by electrophoresis, to isolate the desiredfragment(s).

The fragments may be labeled by conventional techniques. The nature ofthe label and degree of labeling are not critical, depending on thesensitivity that is desired. Radionuclides, fluorochromes and enzymes(e.g., biotin) may be used. ³² p is a preferred label. The fragments maybe labeled with ³² p by nick translation with a α-³² P-dNTP or other ³²p labeling procedure.

HYBRIDIZATION ASSAYS USING LABELED DNA DERIVED FROM ARV

Samples of lymphocytes, such as peripheral blood lymphocytes (PBL) orsplenocytes from patients may be used in the hybridization assay. PBLsare preferred because they are convenient to obtain.

Conventional nucleic acid hybridization techniques are used in theassay. The cells may be applied to the support, such as a nitrocellulosefilter, and lysed in place with a cytolytic agent such as 0.1 to 1 MNaOH to liberate nucleic acid. Alternatively, the cells may be lysedbeforehand and the lysate applied to the support. If the lysing agent isnot one that also denatures the liberated nucleic acids, the lysate isfurther treated with a nucleic acid denaturant such as DNAase/RNAase.Following denaturation the denaturant is neutralized with buffer. Thedenatured (single-stranded) nucleic acids are then immobilized (fixed)on the support. When the support is a nitrocellulose filter, fixation isaccomplished by treating the denatured nucleic acid-bearing filter witha high salt concentration (2-4 M NaCl) buffer and the filter is thendried at elevated temperatures, typically 70°-80° C., for several hours.

The single-stranded nucleic acid fixed on the support is then hybridizedwith the probe. Before hybridization it may be desirable to pretreat thefilter with denatured nonviral-derived DNA (i.e., an ssDNA that will nothybridize to the probe). Such pretreatment saturates binding sites onthe filter and prevents nonspecific binding of the probe to the filter.This technique is well known and is commonly called "prehybridization".Hybridization with the probe is achieved by transferring the filter to aprobe-containing hybridization solution. The concentration of the probein the solution may depend on the size of the probe. Usually the probeis used in excess. The hybridization temperature will depend upon thenature of the hybridization medium (the medium may affect the nucleicacid melting point). Optimum hybridization is generally considered tooccur at the melting point of the nucleic acid minus approximately 20°C. In an aqueous medium, the temperature will normally be about 80° C.to 90° C. The duration of the incubation should be sufficient to allowsubstantially complete hybridization to occur. The hybridization willusually be complete within 12 to 24 hrs.

After the hybridization the support is washed with buffer (variousdegrees of stringency are used in the art) that normally contains asmall amount of surfactant to remove unbound probe. After washing thepresence of labeled duplexes on the filter is detected. The mode ofdetection will depend on the nature of the label. When the label is ³²p, conventional β-emission detectors, e.g., scintillation counters, areused.

As an alternative to the filter hybridization assay described above,cytohybridization procedures may be used. In cytohybridizations thecells are fixed on a support such as a slide or cover slip and treatedwith alkali or heat to partially denature the cellular DNA. The labeledprobe is then applied directly to the cells under conditions similar tothose used in the filter hybridization.

PREPARATION OF ANTI-ARV ANTIBODIES

Sera containing high titers of anti-ARV antibodies may be made byimmunizing laboratory animals, e.g., rabbits, mice, guinea pigs, withARV antigen. The antigen may be in the form of ARV particles, infectedHUT-78 cells, or immunogenic ARV proteins. The animal will typically beinoculated and boosted at least once with the antigen. Serum iscollected from the host and its anti-ARV content determined byimmunoassay using the original inoculant as antigen.

Anti-ARV monoclonal antibodies may be made by conventional somatic cellhybridization techniques using Ig-producing cells or precursors(lymphocytes, splenocytes) from immunized animals or humans having hightiters of anti-ARV antibody and available tumor cell lines as fusionpartners. Mice and murine myelomas are preferred because of theiravailability. These antibodies may be used to identify and isolateimmunogenic ARV proteins by conventional techniques such as immunoassayand affinity chromatography. The proteins may be coupled (immobilized)to solid phases (e.g., beads made or polyvinylchloride, polystyrene,latex, etc.) for use in immunoassays. Purified proteins may be partiallysequenced and the sequence information used to deduce nucleotidesequences for making probes to identify the ARV gene(s) that encode theprotein(s). Once identified the genes may be isolated and cloned intoexpression vectors. These vectors may be used to transform competenthosts to produce transformants that are capable of producing the viralprotein.

AIDS VACCINES

Vaccines may be made using inactivated forms of ARV virus or,preferably, ARV vaccine subunits (e.g., immunogenic ARV polypeptides orimmunogenic fragments thereof). As used in this context the term"immunocenic" is intended to mean a polypeptide which upon introductioninto humans causes an immune response in the recipient that results inthe production of virus-neutralizing antibody. Depending upon the sizeof the polypeptides it may be desirable to conjugate them to carrierproteins such as ovalbumin or serum albumin prior to theiradministration. The vaccine composition will include an immunogenicamount of the polypeptide, a parenteral vehicle, and, optionally, animmunopotentiator. The vaccine will be administered parenterally.

The following example further illustrates various aspects of theinvention. This example is not intended to limit the invention in anymanner.

IDENTIFICATION AND ISOLATION OF ARV

Peripheral mononuclear cell (PMC) cultures were established from 10-30ml of heparinized blood from individuals seen at the Kaposi's SarcomaClinic, University of California, San Francisco (UCSF) or the AIDSClinic, San Francisco General Hospital, USCF. All patients and most ofthe clinically healthy individuals were randomly selected and had livedin San Francisco for at least 2 years. The PMC were separated onFicoll-Hypaque (F/H) gradients. After washing, cells were plated atapproximately 2×10⁶ cells/ml in RPMI 1640 containing 10% fetal bovineserum and antibiotics (100 u/ml penicillin, 100 μg/ml of streptomycin).To this medium was added interleukin-2 (IL-2) (0.5 μg/ml) (MeloyLaboratories, Springfield, Va.) and polybrene (1 μg/ml). At initiationof the cultures, approximately 2.5 μg/ml of phytohemagglutinin (PHA)(Wellcome Reagents, Ltd., England) was added. Some cultures alsoreceived 10⁻⁵ M 2-mercaptoethanol and sheep anti-alpha interferon seraprovided by the National Institutes of Health (Lot #61220), Dr. KariCantell, Helsinki, or Dr. Francoise Barre-Sinoussi, Paris. Theseanti-interferon sera were used at a dilution which neutralized 700-1000units of alpha interferon/mi/culture. The culture supernatants wereroutinely assayed for Mg⁺⁺ -dependent reverse transcriptase (RT)activity every 3-6 days using the following procedure.

Culture medium (1-5 ml) from PMC was spun at 40,000 rpm, 4° C., 45 min,in a Beckman SW41 rotor. The pellets were assayed in a 50 μl reactioncontaining: 40 mM Tris-HCl pH 7.8; 60 mM KCl; 2.2 mM dithiothreitol; 10mM MgCl₂ ; 0.1% Triton X-100; 30 μCi of ³ H!-TTP (spec. act. 78 Ci/mM)and 50 μg/ml of poly(rA)oligo(dT) (PL Biochemicals). Samples wereincubated at 0° C. for 15 min; the reaction was then run for 1 hr, 37°C., and stopped with 4 ml of 5% trichloroacetic acid/0.005 MNa-pyrophosphate/0.5 N HCl. Precipitates were collected on Whatman GF/Afilters, washed, dried and counted in an LKB liquid scintillationcounter. FIG. 1 shows the graph of the results for a representativeculture. As shown, a high level of RT activity was noted in the cultureon the 15th day. On day 22 (arrow), when the RT level was low, thesupernatant fluid was removed and inoculated onto fresh human PMCstimulated 3 days before with PHA. Supernatants from this culture,within six days, contained RT activity at levels of 650,000 cpm/ml andyielded the virus isolate, ARV-2.

The results of RT activity tests on all PMCs tested are reported inTable 1.

                  TABLE 1    ______________________________________    Detection of ARV in AIDS Patients    and Other Individuals from San Francisco                      Number Positive    Diagnosis         Number Tested                                  % Positive    ______________________________________    AIDS with Kaposi's sarcoma                      22/41       53.6    AIDS with opportunistic    infection         0/4         0    Lymphadenopathy Syndrome                       5/10       50.0    Sexual male contacts of    AIDS patients*     3/14       21.4    Clinically healthy homo-    sexual men        2/9         22.2    Clinically healthy hetero-    sexual individuals                       1/23        4.0    ______________________________________     *Steady sexual contact with a patient for at least 6 months before the     onset of his disease.

As reported, in cultures prepared from 41 homosexual AIDS patients, 22were positive for Mg⁺⁺ --dependent (RT) activity. The viruses were foundprimarily in PMC of patients in the early stages of disease. The RTactivity was detected in the PMC usually within the first two weeks ofculture with the peak of activity observed by 12-16 days (FIG. 1). Whenbone marrow aspirates were cultured, 3 out of 9 cultures from AIDSpatients showed evidence of retroviruses. With some cell cultures, theuse of anti-interferon antiserum helped demonstrate the presence ofvirus. When RT activity diminished in the cultures after 2-3 weeks, theaddition of fresh lymphocytes from normal donors would sometimesreestablish RT activity. Supernatant fluids from positive cultures (somestored at -70° C. for over 3 months) also induced RT activity inuninfected fresh lymphocyte cultures. Mg⁺⁺ -dependent RT activity wasalso observed repeatedly in PMC cultures from patients withlymphadenopathy syndrome (LAS), steady sexual male contacts of AIDSpatients, clinically healthy homosexual men, and in one healthy youngheterosexual man.

The viruses detected in seven of the PMC cultures were grown in hightiter and had similar characteristics. ARV-2, mentioned previously, wasrecovered within 2 weeks directly from the PMC of a patientapproximately one month prior to the onset of AIDS. ARV were isolatedfrom subsequent cultures taken two and six months later, after the onsetof AIDS. Multiple time spaced samplings from 5 out of 6 other patientswith AIDS have also yielded retroviruses.

FURTHER CHARACTERIZATION

A. Density of ARV Particles

Supernatant from a culture of ARV-infected PMC was concentrated bycentrifugation in a Beckman SW55 rotor (45,000 rpm, 4° C., 30 min). Thepellet was resuspended in 100 μl of TNE (10 mM Tris-HCl pH 8.0; 100 mMNaCl; 1 mM EDTA pH 8.0) and layered on a 20%-60% (by weight) sucrosegradient in TNE and centrifuged in a Beckman SW55 rotor (35,000 rpm, 4°C., 16 hr). 200 μl fractions were collected from the top and assayed forRT activity. Density of sucrose was determined by refractive indexmeasurements. The RT activity of the viruses was associated withparticles banding at 1:14 to 1:16 g/ml (FIG. 2).

B. Mg⁺⁺ Preference

The viral enzyme, using poly(rA):oligo dT or poly(rC):oligo dG astemplate primers, had up to an eight-fold cation preference for Mg⁺⁺over Mn⁺⁺. With Mg⁺⁺, RT levels higher than 3.5×10⁶ cpm/ml of culturesupernatant could be reached.

C. Effect on Multinucleated Cells

The isolated viruses induced multinucleated cells in lymphocytecultures. They did not immortalize the cells.

D. Morphology

EM analyses of thin sections of PMCs producing ARV were made. Cells werefixed in glutaraldehyde, washed and postfixed in osmium tetroxide andembedded in Araldite. Thin sections were stained in uranyl acetate andlead citrate. Particles with characeristic type D retrovirus morphologywere detected. Budding forms showed early features of both type C andtype D particles, but only mature type D particles were observed in thecultures.

E. Serology

Cell cultures infected with the seven ARV isolates were studied forcross-reactivity with anti-HTLV-I and anti-LAV antibodies by standardindirect immunofluorescence assays (IFA) using fluoresein-labeled goatanti-mouse IgG or goat anti-human IgG. In these assays, cells werespotted onto class slides, air dried and fixed in cold acetone for 15min. An HTLV p19 ten imonoclonal antibody provided by Drs. Robert-Guroffand Gallo, NIH, and an ATLV p19/p28 monoclonal antibody provided by Dr.Y. Hinuma, Kyoto, Japan were used. For detection of LAV, human serum(BRU) provided by Dr. Barre-Sinoussi, Paris was used. Only the anti-LAVantibody reacted with the cells with up to 20% of the cells exhibitingcross-reactivity.

Sera (heated at 56° C., 30 min. used at 1:10 dilution) from AIDSpatients and other individuals from San Francisco were tested for thepresence of anti-ARV antibodies by a similar IFA using a HUT-78 linecontaining 40% of the cells infected with ARV-2. The results of thesetests are reported in Table 2 below.

                  TABLE 2    ______________________________________    Antibodies to ARV in Sera From AIDS    Patients and Other Individuals from San Francisco#    Diagnosis              Anti-ARV    ______________________________________    AIDS with Kaposi's sarcoma (KS)                           59*/67 (88%)    AIDS with opportunistic infection                            18/18 (100%)    Lymphadenopathy Syndrome (LAS)                           22*/27 (81%)    Sexual male contacts of AIDS patients                            11/12 (92%)    Clinically healthy homosexual men                           27*/47 (57%)    Clinically healthy heterosexual individuals                           0*/53 (0%)    ______________________________________     #Figure expressed as number positive/number tested (% positive)     *When antibody negative sera were tested at a 1:5 dilution against the     ARVinfected cells, 8/9 AIDS, 3/5 LAS patients, and 2/12 healthy homosexua     controls showed reactivity; none of the sera from healthy heterosexual     individuals was positive at this dilution.

These results indicate a very high prevalence of antibodies to ARV insera from patients with AIDS and LAS, and in sera from steady sexualcontacts and healthy homosexual men. When the antibody negative serafrom patients with AIDS and LAS were tested at a lower dilution, all but2 reacted with the ARV-infected cells. No antibodies were detected inthe sera of randomly selected healthy heterosexual men from SanFrancisco. All available sera of individuals from whom ARVs wereisolated had antibodies to the virus. Some patients' sera titered 1:640.The high frequency of antibodies in healthy homosexuals may reflect abias from some who volunteered for these studies. Nevertheless, theseresults indicate the very close association of ARV with AIDS and LAS,its widespread presence in the homosexual community, and the greaterdetection of antibodies to ARV than infectious virus. The data supportthe contention that LAS is related to AIDS.

Modifications to the above-described modes for carrying out theinvention that are obvious to those of skill in medicine (particularlyinfectious diseases), immunology, biochemistry, genetic engineering andrelated fields are intended to be within the scope of the followingclaims.

What is claimed is:
 1. A method of preparing antibodies to ARV-2, saidmethod comprising:a) immunizing a host animal with an immunogenic amountof an ARV-2 antigen; and b) isolating said antibodies from animmunoglobulin-containing body fluid collected from said host animal. 2.An isolated, immunogenic protein from ARV-2 virus. wherein said ARV-2virus infects the human T cell line ATCC CRL
 8597. 3. The protein ofclaim 2, wherein said ARV-2 virus has a characteristic of being of typeD morphology by electron microscopy.
 4. The protein of claim 2, whereinsaid ARV-2 virus has a characteristic of having Mg⁺⁺ dependent reversetranscriptase activity.
 5. The protein of claim 2, wherein said ARV-2virus has a characteristic of being capable of inducing humanmultinucleated cells without immortalizing the cells.
 6. The protein ofclaim 2, wherein said ARV-2 virus is capable of replicating in HUT-78human T cells.
 7. The protein of claim 2, wherein said ARV-2virusfurther has a characteristic of inducing viral protein in HUT-78 human Tcells which protein is recognized by immunoglobulins specific to AIDSpatients.
 8. An isolated, immunogenic protein from ARV-2, wherein saidARV-2 virus has characteristics selected from the group ofcharacteristics consisting of:being of type D morphology by electronmicroscopy; having Mg⁺⁺ dependent reverse transcriptase activity; beingcapable of inducing human multinucleated cells without immortalizing thecells; capable of replicating in HUT-78 human T cells; and inducingviral protein in HUT-78 human T cells which protein is recognized byimmunoglobulins specific to AIDS patients.
 9. The protein of claim 8,wherein said ARV-2 is a strain of ARV-2 infecting the human T cell lineATCC CRL
 8597. 10. An isolated, immunogenic protein from anAIDS-associated retrovirus ARV-2, said retrovirus having all of theidentifying characteristics of the ARV-2 retrovirus that has infectedthe human T cell line, ATCC CRL
 8597. 11. The method according to claim1, wherein said ARV-2 antigen is ARV-2 viral particles.
 12. The methodaccording to claim 1, wherein said ARV-2 antigen is infected HUT-78cells deposited as ATCC CRL
 8597. 13. The method according to claim 1,wherein said ARV-2 antigen is an immunogenic ARV-2 protein.